In a preparation of linker chain L1 and 24 kD, an impurity was recently identified as a possible additional linker chain L4. The amino acid sequence of L4 was determined. The molecular mass of L4 was measured by matrix-assisted laser desorption mass spectrometry. A small, but significant deviation between calculated and measured mass of L4 found and is currently being investigated. The sugar portion of linker chains of earthworm hemoglobins is possibly involved in the assemblage of the macromolecular structure of the intact molecule. The glycosylation of the different linker chains of earthworm hemoglobin is still controversial. Matrix-assisted laser desorption mass spectrometry is being used to monitor the deglycosylation of earthworm hemoglobin linker chains. Molecular weight measurements of linker chains L1 and L2 revealed that the enzymatic deglycosylation by N-glycanase was incomplete.